Dengue Fever (DF) and Dengue Hemorrhagic Fever (DHF) are significant public health problems internationally, and caused by four antigenically distinct serotypes of dengue virus (DV1-4). Approximately 36 million cases of DF and 2.1 million cases of DHF occur annually and 2.5-3.5 billion of the world population are at risk of transmission of DF. Although patients who have recovered from DV infection are immune to re-challenge with the same serotype, secondary infection with a different DV serotype can lead to increased risk of DHF and dengue shock syndrome (DSS). The DV genome consists of structural and non-structural proteins with DV serotypes 1-4 having approximately 60%-74% sequence homology in the E gene, which can induce cross-reacting antibodies.
Considerable effort has been devoted to the development of effective vaccines against DV. Live attenuated viruses, inactivated viruses, recombinant proteins, chimeric viruses, DNA vaccines, and synthetic peptides are being evaluated in the clinic. Due to the lack of an animal model or in vitro markers for attenuation in humans, chimeric vaccines with two or less dengue antigens, which results in limited T cell immunity, are being pursued. While antibodies against one serotype can be neutralizing and protective, risk of DHF after exposure to different serotypes has been observed. Early vaccine studies also demonstrated T cell responses to DV, but they were largely DV serotype specific. This may suggest that the level of presentation of MHC class I (MHCI) and class II (MHCII) antigens differs among serotypes. Beneficial effects of the vaccine-induced Th1 response further underscore the significance of the T cell response in vaccine development. The weight of evidence suggests that a useful Dengue virus vaccine will require both B- and T-cell responses to not only successfully protect against infection by each of the four serotypes, but also against the complications of antibody dependent enhancement (ADE).
The present disclosure involves peptides that are associated with the HLA-A2, HLA-A24, or HLA-B7 molecules, HLA-A2 supertypes, HLA-A24 supertypes, and HLA-B7 supertypes. A supertype is a group of HLA molecules that present at least one shared epitope. The present disclosure involves peptides that are associated with HLA molecules, and with the genes and proteins from which these peptides are derived.
Three different methodologies have typically been used for identifying the peptides that are recognized by CTLs in infectious disease field. These are: (1) the genetic method; (2) motif analysis; (3) the immunological and analytical chemistry methods or the Immunoproteomics method.
The genetic method is an approach in which progressively smaller subsets of cDNA libraries from diseased cells are transfected into cells that express the appropriate MHC molecule but not the disease-specific epitope. The molecular clones encoding T cell epitopes are identified by their ability to reconstitute disease specific T cell recognition of transfected cells. The exact T cell epitope is then identified by a combination of molecular subcloning and the use of synthetic peptides based on the predicted amino acid sequence. Such methods, however, are susceptible to inadvertent identification of cross-reacting peptides, and are not capable of identifying important post-translational modifications.
Motif analysis involves scanning a protein for peptides containing known class I MHC binding motifs, followed by synthesis and assay of the predicted peptides for their ability to be recognized by disease-specific CTL. This approach requires prior knowledge of the protein from which the peptides are derived and widely used in virus or bacterial infection field. This approach is also greatly hampered by the fact that not all of the predicted peptide epitopes are presented on the surface of a cell (Yewdell, J. W. and Bennink, J. R., Ann. Rev. Immunol., 17:51-88, (1999)), thus additional extensive experimentation is required to determine which of the predicted epitopes is useful.
Immunoproteomics method involves a combination of cellular immunology and mass spectrometry. This approach involves the actual identification of endogenous CTL epitopes present on the cell surface by sequencing the naturally occurring peptides associated with class I MHC molecules. In this approach, cells are first lysed in a detergent solution, the peptides associated with the class I MHC molecules are purified, and the peptides are fractionated by high performance liquid chromatography (HPLC). Peptide sequencing is readily performed by tandem mass spectrometry (Henderson, R. A. et al., Proc. Natl. Acad. Sci. U.S.A, 90:10275-10279, (1993).
A number of recent reports for different types of virus infections provide evidence that CTL specific for epitopes that are naturally processed and presented by infected cells have markedly greater impact on the control of virus replication. Undoubtedly, CTLs have been shown to play an important role in the elimination of dengue virus-infected cells. Thus, identification of antigenic peptides that are presented by infected cells and recognized by epitope-specific CTLs may suggest new ways to suppress viral replication and prevent persistent infection. Multiple peptides from conserved regions of dengue virus may prove essential in the development of a universally immunogenic vaccine. In recent years, several MHC class I specific peptides have been reported by the screening of algorithm-predicted T-cell epitopes using T cells from individuals participating in experimental DV vaccine trials as well as those infected with DV. However, these peptides were not subsequently investigated nor determined to be presented by DV infected cells.
Little is known about cross serotype conserved T cell epitopes that are immunologically relevant in eliciting an effective T cell response to the four DV serotypes. Several groups have attempted to identify T cell epitopes by either motif prediction of MHC binding peptides from Dengue proteins, or by screening overlapping peptides from structural and nonstructural Dengue proteins. Screening PBMCs from individuals in a DV vaccine trial and DV-infected patients using a panel of algorithm-derived peptide sequences identified a few DV serotype specific T cell epitopes. However, a comprehensive analysis of naturally presented epitopes on infected cells has never been undertaken or reported.